The long-term goal of this project is to elucidate the events that cause mitochondrial dysfunction in ischemia/reperfusion- and toxicant-induced acute renal failure (ARF), and to identify a therapeutic approach that prevents the mitochondrial dysfunction and reduces ARF. The role of mitochondrial dysfunction and disruption of Ca 2+ homeostasis in renal cell injury and death has been demonstrated in numerous models of ARF and nephrotoxicity. The importance of calpains (Ca2+-activated neutral cysteine proteases) in renal proximal tubule cellular (RPTC) injury and death produced by hypoxia/reoxygenation and toxicants has been shown using calpain inhibitors. In particular, two dissimilar calpain inhibitors not only blocked hypoxia/reoxygenation RPTC death, but also blocked the mitochondrial dysfunction and promoted the recovery of respiration during reoxygenation. These results strongly support a key role for calpains in mitochondrial dysfunction. The above experiments showing calpain inhibitor protection of mitochondrial function in RPTC, suggest that mitochondria may contain a calpain. In a number of diverse preliminary experiments using isolated renal cortical mitochondria (RCM) we have obtained additional evidence of a novel mitochondrial calpain that is responsible for mitochondrial dysfunction. These data resulted in the hypothesis that mitochondrial Ca 2+- uptake leads to the activation of a mitochondrial calpain, which causes the mitochondrial dysfunction and ultimately results in RPTC death and ARF. The specific aims of this application are: Specific Aim I: Identify and characterize the mitochondrial calpain and examine its regulation in isolated RCM and RPTC. Specific Aim II: Elucidate the mechanism of mitochondrial calpain-mediated mitochondrial dysfunction in RPTC and isolated RCM, and identify the mitochondrial protein targets of mitochondrial calpain. Specific Aim III: Determine the effectiveness of currently described calpain inhibitors on mitochondrial calpain and develop new specific inhibitors of mitochondrial calpain using novel, non-natural amino acid analogues and determine their effectiveness in RPTC and isolated RCM. Specific Aim IV: Determine the efficacy of current and/or developed calpain inhibitors in an in vivo model of mitochondrial dysfunction and ARF. Completion of these Specific Aims will add significantly to our basic understanding of cell injury and death, particularly events mediating mitochondrial dysfunction. Further, we will identify a mitochondrial calpain and develop novel calpain inhibitors, including those that are mitochondrial calpain specific. Ultimately, these studies may lead to the development of therapeutic agents that improve clinical outcomes in patients with ARF. [unreadable] [unreadable]